期刊
NATURE PROTOCOLS
卷 9, 期 10, 页码 2411-2424出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.154
关键词
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资金
- National Heart, Lung, and Blood Institute of the US National Institutes of Health (NIH) as a Program of Excellence in Nanotechnology [HHSN268201000043C]
- NIH [DP3DK094346]
- National Science Foundation Science and Technology Centers (NTC) [CBET-0939511]
- Atlanta Clinical and Translational Science Institute (ACTSI) (Clinical and Translational Science Award program) [UL1 RR025008]
- Atlanta Clinical and Translational Science Institute (ACTSI) (NIH)
- Atlanta Clinical and Translational Science Institute (ACTSI) (National Center for Research Resources)
- American Heart Association
Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with similar to 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.
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